A twisted base? The role of arginine in enzyme-catalyzed proton abstractions

Arginine residues are generally considered poor candidates for the role of general bases because they are predominantly protonated at physiological pH. Nonetheless, Arg residues have recently emerged as general bases in several enzymes: IMP dehydrogenase, pectate/pectin lyases, fumarate reductase, and l-aspartate oxidase. The experimental evidence suggesting this mechanistic function is reviewed. Although these enzymes have several different folds and distinct evolutionary origins, a common structural motif is found where the critical Arg residue is solvent accessible and adjacent to carboxylate groups. The chemistry of the guanidine group suggests unique strategies to lower the pK(a) of Arg. Lastly, the presumption that general bases must be predominantly deprotonated is revisited.     

Characterization of the putative native and recombinant rat sterol transporter Niemann-Pick C1 Like 1 (NPC1L1) protein

The exact mechanistic pathway of cholesterol absorption in the jejunum of the small intestines is a poorly understood process. Recently, a relatively novel gene, Niemann-Pick C1 Like 1 (NPC1L1), was identified as being critical for intestinal sterol absorption in a pathway which is sensitive to sterol absorption inhibitors such as ezetimibe. NPC1L1 is a multi-transmembrane protein, with a putative sterol sensing domain. Very little else is known about the NPC1L1 protein. In this report, we characterize the native and recombinant rat NPC1L1 protein. We show that NPC1L1 is a 145 kDa membrane protein, enriched in the brush border membrane of the intestinal enterocyte and is highly glycosylated. In addition, sequential detergent extraction of enterocytes result in highly enriched preparations of NPC1L1. An engineered Flag epitope tagged rat NPC1L1 cDNA was expressed as recombinant protein in CHO cells and demonstrated cell surface expression, similar to the native rat protein. These biochemical data indicate that NPC1L1 exists as a predominantly cell surface membrane expressed protein, consistent with its proposed role as the putative intestinal sterol transporter.     

IMP dehydrogenase: structural schizophrenia and an unusual base

Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additional conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.     

Agrobacterium-mediated transient transformation of marigold (Tagetes erecta)

Hypocotyls, roots, leaf sections and shoot tips from Tagetes erecta plantlets were inoculated with Agrobacterium tumefaciens, harboring the binary vector pCAMBIA2301, containing the β-glucuronidase gene. Histochemical GUS assays of infected tissues showed transient gus gene expression after 3 days.     

Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.     

Conformational distributions of protease-serpin complexes: a partially translocated complex

Serpins regulate serine proteases by forming metastable covalent complexes with their targets. The protease docks with the serpin and cleaves the serpin's reactive center loop (RCL) forming an acylenzyme intermediate. Cleavage triggers insertion of the RCL into beta sheet A, translocating the attached protease approximately 70 A and disrupting the protease active site, trapping the acylenzyme intermediate. Using single-pair F?rster resonance energy transfer (spFRET), we have measured the conformational distributions of trypsin and alpha(1)-proteinase inhibitor (alpha(1)PI) covalent complexes. Bovine trypsin (BTryp) complexes display a single set of conformations consistent with the full translocation of BTryp (E(full)I*). However, the range of spFRET efficiencies is large, suggesting that the region around the trypsin label is mobile. Most complexes between alpha(1)PI variants and the more stable rat trypsin (RTryp) also show a single set of conformations, but the conformational distribution is narrower, indicating less disruption of RTryp. Surprisingly, RTryp complexes containing alpha(1)PI labeled at Cys232 with a cationic fluorophore display two equally populated conformations, E(full)I* and a conformation in which RTryp is only partially translocated (E(part)I*). Destabilizing the RTryp active site, by substituting Ala for Ile16, increases the E(full)I* population. Thus, interactions between anionic RTryp and cationic dyes likely exert a restraining force on alpha(1)PI, increasing the energy needed to translocate trypsin, and this force can be counteracted by active site destabilization. These results highlight the role of protease stability in determining the conformational distributions of protease-serpin covalent complexes and show that full translocation is not required for the formation of metastable complexes.     

Structure-activity relationship study of selective benzimidazole-based inhibitors of Cryptosporidium parvum IMPDH

Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The Cryptosporidium parvum and Cryptosporidium hominis genomes indicate that the only route to guanine nucleotides is via inosine 5'-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazole-based inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure-activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro.     

The dynamic determinants of reaction specificity in the IMPDH/GMPR family of (β/α)(8) barrel enzymes

The inosine monophosphate dehydrogenase (IMPDH)/guanosine monophosphate reductase (GMPR) family of (β/α)(8) enzymes presents an excellent opportunity to investigate how subtle changes in enzyme structure change reaction specificity. IMPDH and GMPR bind the same ligands with similar affinities and share a common set of catalytic residues. Both enzymes catalyze a hydride transfer reaction involving a nicotinamide cofactor hydride, and both reactions proceed via the same covalent intermediate. In the case of IMPDH, this intermediate reacts with water, while in GMPR it reacts with ammonia. In both cases, the two chemical transformations are separated by a conformational change. In IMPDH, the conformational change involves a mobile protein flap while in GMPR, the cofactor moves. Thus reaction specificity is controlled by differences in dynamics, which in turn are controlled by residues outside the active site. These findings have some intriguing implications for the evolution of the IMPDH/GMPR family.     

Meta-analysis of genome-wide scans for total body BMD in children and adults reveals allelic heterogeneity and age-specific effects at the WNT16 locus

To identify genetic loci influencing bone accrual, we performed a genome-wide association scan for total-body bone mineral density (TB-BMD) variation in 2,660 children of different ethnicities. We discovered variants in 7q31.31 associated with BMD measurements, with the lowest P = 4.1 × 10(-11) observed for rs917727 with minor allele frequency of 0.37. We sought replication for all SNPs located ± 500 kb from rs917727 in 11,052 additional individuals from five independent studies including children and adults, together with de novo genotyping of rs3801387 (in perfect linkage disequilibrium (LD) with rs917727) in 1,014 mothers of children from the discovery cohort. The top signal mapping in the surroundings of WNT16 was replicated across studies with a meta-analysis P = 2.6 × 10(-31) and an effect size explaining between 0.6%-1.8% of TB-BMD variance. Conditional analyses on this signal revealed a secondary signal for total body BMD (P = 1.42 × 10(-10)) for rs4609139 and mapping to C7orf58. We also examined the genomic region for association with skull BMD to test if the associations were independent of skeletal loading. We identified two signals influencing skull BMD variation, including rs917727 (P = 1.9 × 10(-16)) and rs7801723 (P = 8.9 × 10(-28)), also mapping to C7orf58 (r(2) = 0.50 with rs4609139). Wnt16 knockout (KO) mice with reduced total body BMD and gene expression profiles in human bone biopsies support a role of C7orf58 and WNT16 on the BMD phenotypes observed at the human population level. In summary, we detected two independent signals influencing total body and skull BMD variation in children and adults, thus demonstrating the presence of allelic heterogeneity at the WNT16 locus. One of the skull BMD signals mapping to C7orf58 is mostly driven by children, suggesting temporal determination on peak bone mass acquisition. Our life-course approach postulates that these genetic effects influencing peak bone mass accrual may impact the risk of osteoporosis later in life.     

Retinal isoforms of inosine 5'-monophosphate dehydrogenase type 1 are poor nucleic acid binding proteins

The RP 10 form of autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in the widely expressed protein inosine 5′-monophosphate dehydrogenase type 1 (IMPDH1). These mutations have no effect on the enzymatic activity of IMPDH1, but do perturb the association of IMPDH1 with nucleic acids. Two newly discovered retinal-specific isoforms, IMPDH1(546) and IMPDH1(595), may provide the key to the photoreceptor specificity of disease [S.J. Bowne, Q. Liu, L.S. Sullivan, J. Zhu, C.J. Spellicy, C.B. Rickman, E.A. Pierce, S.P. Daiger, Invest. Ophthalmol. Vis. Sci. 47 (2006) 3754-3765]. Here we express and characterize the normal IMPDH1(546) and IMPDH1(595), together with their adRP-linked variants, D226N. The enzymatic activity of the purified IMPDH1(546), IMPDH1(595) and the D226N variants is indistinguishable from the canonical form. The intracellular distribution of IMPDH1(546) and IMPDH1(595) is also similar to the canonical IMPDH1 and unaffected by the D226N mutation. However, unlike the canonical IMPDH1, the retinal specific isoforms do not bind significant fractions of a random pool of oligonucleotides. This observation indicates that the C-terminal extension unique to the retinal isoforms blocks the nucleic acid binding site of IMPDH1, and thus uniquely regulates protein function within photoreceptors.